The current evidence indicates that ACTH regulates adrenal steroidogenesis by affecting pregnenolone synthesis in the mitrochondria via a cAMP-dependent protein kinase and hence involves the phosphorylation of a protein(s). A rapidly turning-over protein also is involved since inhibitors of protein synthesis prevent the acute effect of ACTH. The level of phosphorylation of a protein will be determined by the balance between the rates of phosphorylation and dephosphorylation. Recent findings suggest that the dephosphorylation reaction may be determining in some cases. Two classes of heat-stable protein inhibitors of phosphoprotein phosphatases have been found. One class, inhibitor-1, requires phosphorylation by a cAMP-dependent protein kinase to be active, while the other class, inhibitor-2, does not. The level of phosphorylation of inhibitor-1 responds to epinephrine and insulin, in vivo. There also appears to be a relationship between the two types of inhibitors in their action on protein phosphatases. It is proposed to investigate the possible role of these inhibitors in the response of the adrenal to ACTH and other aspects of phosphoprotein dephosphorylation in the synthesis of pregnenolone in the mitochondria. This will involve: (1) the determination of the relationship of steroidogenesis to the levels of phosphorylation of inhibitor-1 and the activity of inhibitor-2 in the total tissue and in the subcellular fractions of adrenals incubated with and without ACTH in the absence and presence of inhibitors of protein synthesis; (2) the determinatio of the relationships between pregnenolone synthesis and in vitro induced changes in the levels of phosphorylation in mitochondria obtained from adrenals incubated with ACTH; (3) an investigation to assure that no, or a minimum of, artifacts of phosphorylation or dephosphorylation are introduced into phosphoproteins during homogenization and subcellular fractionation. The investigations described carry the potential for the identification of phosphoproteins involved in the action of ACTH. This may be applicable in investigations of adrenal manfunction in disease.